Anti-microbial T cell responses play a major role in determining the outcome of infection. Chronic infections are often distinguished by T cell responses that are not able to fully eliminate the pathogen. The mechanisms that explain this failure of T cell effector responses are only beginning to be understood. During the current funding period, we have used the lymphocytic choriomeningitis virus (LCMV) model to investigate how the PD-1 pathway regulates T cell responses during chronic infection. We have found that the PD-1 pathway has multifaceted roles in protective immunity. We discovered that in addition to PD-L1, PD-L2 limits responses of exhausted T cells. We also have shown that PD-L1 has distinct roles on hematopoietic and non- hematopoetic cells during chronic infection. However, we lack a mechanistic understanding of PD-L1 and PD-L2 functions on specific cell types. A major goal of this proposal is to elucidate mechanisms by which PD-1 and its ligands regulate CD8 T cell exhaustion, issues of fundamental and translational importance, given the therapeutic promise of this pathway. In addition, we have discovered a new function for the PD-1 pathway in protective immunity. We have identified a role for PD-1 in regulating humoral immunity by inhibiting the generation and function of T follicular regulatory cells. Thus, we will not only address how the PD-1 pathway regulates CD8 T cell exhaustion, but also how it controls humoral immunity. In addition, we will study roles of two novel coinhibitory molecules identified by PPG investigators in anti-microbial immunity: repulsion guidance molecule b (RGMb) and protein C receptor (PROCR). Dr. Freeman (Core B) identified RGMb as a second binding partner for PD-L2. The rescue of T cell exhaustion in PD-L2 deficient mice leads us to study RGMb function during chronic infection. Dr. Kuchroo (PI, Project 3) has identified protein C receptor (PROCR) as a novel coinhibitory molecule; in collaboration we have found that PROCR is highly expressed on exhausted CD8 T cells. We will study if PROCR regulates T cell exhaustion. Our main hypothesis is that PD-1 pathway members and PROCR regulate protective immunity during acute and chronic infections. To test this hypothesis, our Specific Aims are to: 1) Analyze how PD-1 and its ligands regulate humoral immunity during acute mucosal/localized viral infection (influenza) versus systemic infection (LCMV). 2) Investigate roles of the PD-1 and PROCR pathways in controlling exhausted CD8 T cells during chronic LCMV infection. Our goals are to determine how to optimally modulate the PD-1 and PROCR pathways therapeutically in chronic infection, and develop new strategies to promote protective immunity during acute viral infections.